This invention relates to antigenic conjugates of the conserved portion of the lipopolysaccharides of certain gram negative bacteria and to vaccines containing such antigenic conjugates. The conjugates elicit antibodies which exhibit cross reactivity against heterologous strains of gram negative bacteria and the vaccines containing such conjugates induce antibodies which are functional and protective against such gram negative bacterial organisms.
Lipopolysaccharides (LPS) are major surface antigens localized abundantly on the surface of gram negative bacteria. LPS molecules are comprised of: (1) a lipid A portion which consists of a glucosamine disaccharide substituted with phosphates, phosphoethanolamine groups and long chain fatty acids in ester and amide linkages; (2) an inner core portion attached to the lipid A portion by an eight carbon sugar, ketodeoxyoctonoate (KDO), which may be substituted by 1 to 2 additional KDO molecules and by up to 3 heptose moieties; (3) an outer core portion comprising hexoses such as glucose, galactose, N-acetylglucosamine and N-acetylgalactosamine; and (4) an O-specific chain comprising repeating oligo-saccharide units which vary widely among bacterial strains. Polymerization of these repeating units to structures in excess of 60,000 daltons is not uncommon. The LPS molecules can vary extensively at the structural and antigenic level among bacterial strains, although the structure of the inner core is largely conserved among bacterial species. A typical structure of the lipid-A inner core of Salmonella typhimurium LPS is illustrated in FIG. 1. The immune response responsible for the evolution of naturally protective antibodies is considered to arise by natural immunization to this region of the LPS.
In non-enteric pathogens, the LPS structure lacks repeating O-antigen units. Moreover, the complete genetic machinery for assembly of the O-antigen repeating unit appears to be absent in such pathogens. This has led to the designation of these LPS structures as lipooligosaccharides (LOS). There are similarities between LPS and LOS structures in such pathogens. For instance, all of the LPS and LOS structures link the lipid A regions to the cores through the KDO junction. The number of KDO residues can vary from one (e.g., Haemophilus influenzae and Haemophilus ducreyi) to two (e.g., Neisseria meningitidis and Neisseria gonorrhoeae). Recent studies indicate that the branched oliogsaccharides are synthesized separately from the core region and the assembly of the entire LOS structure is completed on the outer side of the cytoplasmic membrane (see Preston, et al., xe2x80x9cThe Lipooligosaccharides of Pathogenic Gram-Negative Bacteriaxe2x80x9d, Critical Reviews In Microbiology, 22:139-180 (1996)).
Accordingly, there is a single core region in such LOS structures without a distinct inner and outer core region. The core structure of these pathogens can vary from species to species and may comprise KDO in the complete absence of heptose (e.g., Moraxella catarrhalis); KDO in the presence of a di-heptose structure (e.g., Neisseria meningitidis and Neisseria gonorrhoeae); or KDO in the presence of a tri-heptose structure (e.g., Haemophilus influenzae and Haemophilus ducreyi). Examples of core structures from Haemophilus and Neisseria are shown in FIG. 2. The oligosaccharide units can extend from each of the heptoses and/or they can be substituted by phosphoethanolamine groups. Typical examples of completed LOS structures of Haemophilus influenzae strain 2019 (see Phillips et al., xe2x80x9cStructural Characterization of the Cell Surface Lipooligosaccharides from a Non-Typable Strain of Haemophilus Influenzae,xe2x80x9d Biochemistry, 31:4515-4526 (1992)) and Neisseria gonorrhoeae strain 1291 (see John et al., xe2x80x9cThe Structural Bases for Pyocin Resistance in Neisseria gonorrhoeae lipooligosaccharides,xe2x80x9d J. Biol. Chem., 266:1903-1911 (1991)) are shown in FIG. 3.
The use of LPS in the development of vaccines is known in the art. It has long been recognized that a specific antibody response directed against the LPS of a particular bacterial pathogen can contribute to protection against that specific strain. It is further known that saccharide structures (e.g., the saccharide portions of LPS) can be conjugated to a carrier protein, so that a vaccine composition containing such a conjugate will elicit the desired T-dependent response. An example of this is the successful glycoconjugate vaccines against bacteria having type-specific capsular saccharides see, Vaccine Design: The Subunit and Adjuvant Approach, Powell, M. F., and Newman, M. J., 673-694 (1995). This category of immune response is the basis for the effectiveness in human infants of a new generation of saccharide-protein conjugate vaccines as discussed in Vaccine Design: The Subunit and Adjuvant Approach, Powell, M. F., and Newman, M. J., 695-718 (1995).
However, since the LPS of heterologous strains of such pathogens demonstrate extensive variation of the outer core saccharide and/or O-specific chain, efforts to generate an antibody response to a number of heterologous strains or heterologous genera of bacteria utilizing a vaccine containing a single LPS have to date been unsuccessful.
In an effort to develop an LPS-based vaccine against Neisseria meningitidis, tetanus toxoid has been conjugated with oligosaccharides isolated from the LPS of a number of Neisseria meningitidis strains (see, Jennings et al., Infect. Immun., 43:407-412 (1984)). However, the antibodies elicited by this conjugate were oligosaccharide specific and exhibited a high degree of serotype specificity.
Verheul et al., Infect. Immun., 61:187-196 (1993), disclose the conjugation of oligosaccharides of meningococcal LPS to either tetanus toxoid or meningococcal outer membrane protein. In mice, the tetanus toxoid conjugates induced oligosaccharide specific antibodies which were not bactericidal against meningococci. The outer membrane proteinxe2x80x94LPS conjugates induced antibodies against the outer membrane protein, but not against LPS. Verheul et al., Infect. Immun., 59:843-851 (1991), also studied the immunogenicity of the conjugates of oligosaccharides of several Neisseria meningitidis strains and tetanus toxoid in rabbits. The results demonstrated that the antibodies elicited are directed only towards the oligosaccharide portion of the LPS which contain the immunotype specific epitopes.
The preparation of oligosaccharides from the LPS of Neisseria meningitidis laboratory adapted wild strain A1 and the subsequent conjugation thereof to tetanus toxoid as the carrier protein was disclosed in Gu et al., Infect. Immun., 61:1873-1880 (1993). The conjugates were immunogenic in mice and rabbits and the majority of the antibodies were directed against immunotype-specific LPS epitopes. Also, the conjugate antisera showed less cross reactivity to different immunotypes of LPS than the LPS antisera. These studies demonstrate that LPS-derived oligosaccharide conjugates induce antibodies to the specific oligosaccharide immunotype. However, there was no evidence that the conjugate induced significant cross reactive antibodies to a common core saccharide structure present in the majority of Neisseria meningitidis strains.
Bhattacharjee et al., J. Infect. Dis., 173:1157-1163 (1996) disclosed the mixture of the LPS from Escherichia coli with the outer membrane protein of Neisseria meningitidis group B, resulting in the formation of unconjugated, non-covalent complexes thereof. It was found that these complexes elicited antibodies which cross reacted with a number of gram negative bacteria. However, no evidence was provided to indicate that these complexes had properties different from other preparations of unconjugated saccharide structures which are known to be incapable of eliciting a T-dependent antibody response which can be boosted upon administration of subsequent doses. Moreover, it is known that such unconjugated saccharides do not elicit an appropriate immune response in infants.
Gu et al., Infect. Immun., 64:4047-4053 (1996) disclose the preparation of conjugates of oligosaccharides from nontypeable Haemophilus influenzae and tetanus toxoid. However, the antisera induced in rabbits demonstrated bactericidal activity against only homologous strains.
Accordingly, there remains a need for antigenic conjugates and vaccines containing such conjugates which effectively induce an immunogenic response, preferably a T-dependent response, to a given species of gram negative bacteria, as well as which exhibit effective cross reactivity to heterologous strains or serotypes of gram negative bacteria within a given genus. Moreover, it would be advantageous for such conjugates and vaccines to elicit antibodies which exhibit cross reactivity to heterologous genera of gram negative bacteria.
The present invention is directed to antigenic conjugates comprising a carrier protein covalently bonded to the conserved LPS portion of a gram negative bacteria, wherein the conserved portion of the LPS comprises at least the conserved inner core and the lipid A portion of the LPS. The conjugate elicits a cross reactive immune response against heterologous strains of gram negative bacteria and preferably, against heterologous genera of gram negative bacteria.
The present invention is further directed to vaccines comprising these antigenic conjugates and methods for immunizing individuals with such vaccines to prevent various diseases caused by gram negative bacteria.